In this R21 exploratory program, novel concepts and systems for in vivo multiphoton microscopic imaging will be explored and demonstrated to enable unprecedented penetration depth into scattering biological tissues. The proposed research consists of two concurrent thrusts: the first involves the exploration of new spectral windows with much reduced tissue scattering for multiphoton microscopy; the second thrust concentrates on the simultaneous focusing of the spatial and temporal components of the illumination field to significantly enhance the signal to background ratio in imaging scattering tissues. These two parallel research components will each improve the penetration depth of current multiphoton microscopy and, depending on the applications involved, can be combined for maximum benefits. Applying these new concepts and techniques, we aim to create a new multiphoton imaging system that offers unprecedented penetration depth, providing new tools for both in vivo detection of cancer and in vivo studies of cancer biology. With its jam-resolution and molecular specificity, in vivo optical imaging holds tremendous potential for non-invasive cancer detection and diagnostics. Because of the strong scattering encountered in most biological tissues, however, optical imaging is now mostly restricted to the superficial layer. This lack of penetration depth severely limits the applicability of optical techniques for cancer research. The objective of this program is to create and develop practical methods for extending the penetration depth, significantly increasing the reach of optical imaging techniques in cancer detection and diagnostics. To accommodate the breadth of this proposal, this program involves close collaboration between the PI and Co-Pi Dr. Warren Zipfel. The three specific aims of the program are: 1. Explore and demonstrate new spectral windows for multiphoton microscopy with reduced tissue scattering. 2. Develop and demonstrate the concept of simultaneous spatial and temporal focusing for enhancing the signal to background ratio in imaging scattering samples. 3. Demonstrate specific aims 1 and 2 for multiphoton microscopic/endoscopic imaging for cancer research. [unreadable] [unreadable]